The combination of alpha-interferon (IFN) and 5-fluorouracil (FUra) has anti-tumor activity in patients with advanced colorectal cancer, and this activity can be duplicated in vitro in human colon carcinoma cell lines. The objectives of this project are to define the biochemical interactions of FUra and IFN which might contribute to the synergistic interaction, and use this information to design novel agents to further modulate the antitumor activity of FUra/IFN in colon and breast cancer. In our preliminary studies, iFN stimulated thymidine phosphorylase (TP) activity (the first enzyme in one pathway in the metabolic activation of FUra) and increased the levels of the FUra active metabolite, FdUMP. Increased TP activity was accompanied by elevated TP mRNA levels. We will determine the nature of the regulatory control of the TP gene by IFN. To test the hypothesis that the induction of TP is critical for the activity of IFN, we have transfected TP into MCF7 breast carcinoma cells. We will compare the sensitivity of the transfected cells to FUra with MCF7 cells in which TP is induced by IFN. We will also determine if the iFN-mediated stimulation is a clinically relevant phenomena by measuring TP in cells from patients treated with iFN. The full potential of the iFN-induced increase in TP activity was not observed in intact cells, however, due to a rate limiting cosubstrate for TP. We will synthesize novel deoxyribonucleosides as potential cosubstrates for TP, and determine their effects on FUra antitumor actions. FUra is incorporated into RNA and misincorporation alters RNA functions. We observed a profound effect of FUra on a mRNA of a gene activated by IFN: the enzyme 2',5'-oligoadenylate synthetase (OAS). The OAS gene transcript is processed to yield two mRNA (1.6 and 1.8 kb). We found that FYra abrogated the formation of the 1.8 kb mRNA, while having no effect on the 1.6 kb mRNA. We will determine the molecular mechanism for the differential effect of FUra, particularly examining alternative hypotheses that effects on OAS mRNA transcription, polyadenylation, splicing, or turnover are involved. We will further define the FUra-sensitive regions (e.g. splicing or poly(A) sites) of the OAS gene by incorporating these into a mammalian expression vector, transfecting into colon carcinoma cells, and measuring the effect of FUra on construct expression.